The activity of many enzymes of physiological importance is known to be regulated by reversible phosphorylation which, in an increasing number of cases, has been observed at multiple sites on one enzyme. Multi-site phosphorylation may be a mechanism for providing a complex pattern of metabolic or other regulation, in response to protein kinase or phosphatase changes, or it may have other functions. For example, multi-site phosphorylation of histone H1 appears to act to modify the structure of chromatin in response to changes in histone H1 kinase activities which phosphorylate multiple sites on histone H1. I will separate the nuclear histone kinases that are active in the period (G2 phase) of cell growth when the phosphorylation pattern of histone H1 changes from the S phase pattern to the mitosis pattern and I will determine the site-specificity of each kinase and the changes in kinase activities that occur in vivo during G2 phase. The properties of the kinases will be correlated with the development in vivo of the mitosis pattern of H1 phosphorylation. The results may give direct insight into the inter-relationships of phosphorylation at multiple sites and will lead to studies of regulation of the kinase(s) involved in mitosis-associated H1 phosphorylation. The procedures will use the naturally synchronous cell cycle in Physarum polycephalum which allows precisely timed studies during G2 phase. Physarum histone H1 will be sequenced and phosphorylation sites will be determined by 32P labelling. A specific substrate and/or inhibitor for the physiologically-important kinase will be developed using either an isolated peptide from histone H1 or a synthetic peptide. Antibodies to the kinase, or a specific kinase-binding peptide, will be used to study localization, synthesis and activation of kinase in vivo, and to study the effects on the cell cycle of modifying kinase activity experimentally in vivo. Eventually, applications in aging, would healing and cancer chemotherapy are envisioned.